Many eye diseases, such as age-related macular degeneration (AMD), diabetic retinopathy (DR) and glaucoma, involve pathological changes in retinal photoreceptors and/or inner retinal neurons. It is well known that different diseases can target different cell types. In principle, physiological modification must occur in diseased cells, before detectable abnormality of retinal morphology. Therefore, functional evaluation of retinal physiology is important for early disease detection and reliable treatment management. At University of Alabama at Birmingham (UAB), we have developed several optical approaches, including functional optical coherence tomography (OCT) and line-scan confocal microscopy, to pursue optophysiological monitoring of physiological activities using transient intrinsic optical signal (IOS) changes correlated with retinal stimulation. We propose here to validate functional IOS mapping of photoreceptor physiology at high resolution (< 50 m). Success of this project can lead to better study and diagnosis of photoreceptor dysfunction associated with eye diseases, including AMD which is the leading cause of severe vision loss and legal blindness. Our preliminary study has revealed IOS abnormalities in one mouse model with inherited photoreceptor degeneration, and in vivo confocal-IOS observation of individual frog photoreceptors have been demonstrated. Fast confocal-IOS imaging promises a high-resolution method for functional examination of photoreceptor physiology, while simultaneously providing information of retinal morphology. However, before clinical deployments of the functional IOS imaging, there are several obstacles: 1) IOS mechanism in the retina is not clear; 2) IOS imaging protocol of rod and cone systems is not developed; 3) clinical relevance between IOS modification and retinal disease is not established. These problems will be tackled in our three specific aims of this project. The first aim is to investigate physiological sources of the IOS. A hybrid confocal-OCT microscope will be employed to dissect the IOS in the photoreceptor and inner retinal cells at sub-cellular resolution. Comparative measurement of normal and aspartate-treated retinas will be used to test physiological pathways of the IOS. The second aim is to develop stimulation protocol for functional IOS mapping of retinal photoreceptors. We anticipate that selective mapping of localized (e.g., 50 m) rod and cone functions can be achieved through quantitative controls of retinal adaptation and stimulation, without the requirement of resolving individual photoreceptors. The third aim is to establish clinical potential of functional IOS imaging. One inherited photoreceptor degeneration model rd10, i.e., C57BL/6J-Pde6brd10, will be used for comparative IOS, electrophysiological, and histological study. Success criterion of this study is to demonstrate that the IOS imaging allows early detection of photoreceptor dysfunction at the time point no later than detectable structural changes in the retina. By the end of this project, ultimate imaging modality (i.e., confocal or OCT) for clinical application will be identified based on quantitative comparison of confocal- and OCT-IOS images.